The temporal and location specific activity of numerous proteins is controlled by myriad posttranslational modifications. Of these exquisite chemical modifications, phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively, regulate a diversity of cellular events from cell division to cell death. The >500 human protein kinases and 147 protein phosphatases in tandem respond to a variety of both intra- and extra-cellular environmental cues and it has been estimated that at least a third of the proteome is capable of being phosphorylated. Not surprisingly, the deregulation of phosphorylation leads to a variety of human diseases including cancer. Despite the importance of phosphorylation driven signaling, decrypting the role of a specific kinase or phosphatase remains enormously challenging. Currently there are almost no uniquely selective small molecules for pharmacological perturbation of native kinases or phosphatases. Prevailing siRNA and other genetic knockdown methods, which provide insight regarding the function of a specific enzyme, cannot afford temporal control and mechanistic details are obscured by compensatory cellular mechanisms. A few elegant and powerful methods have been developed to provide temporal turn-off or turn-on control over a single kinase to study cell biology. However, there are no methods that allow for controlling multiple user-defined kinases and phosphatases that are often implicated in a cell signaling pathway. We hypothesize that this knowledge gap can be addressed by developing an approach that allows for orthogonal small molecule control over split-kinases (Aim 1) as well as split-phosphatases (Aim 2), which we have recently designed. The three proposed aims are designed to provide validated methods with interchangeable parts (Aim 3) for studying signaling. We propose to demonstrate generality, quantify kinetics and substrate specificity, and provide validate methods for studying temporal aspects of specific kinases (Aim 1) and phosphatases (Aim 2) in live cells. These methods will perhaps be particularly relevant for studying the dynamics of cell motility and cell adhesion, which are of fundamental interest as well as central to understanding migration and metastatic pathways in cancer with collaborators.